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PP2A1 binding, cell transducing and apoptotic properties of Vpr(77-92): a new functional domain of HIV-1 Vpr proteins.

Godet AN, Guergnon J, Croset A, Cayla X, Falanga PB, Colle JH, Garcia A - PLoS ONE (2010)

Bottom Line: We also found that the I84P mutation or the IIQ/VTR(83-85) and T89A substitutions in the Vpr(77-92) sequence prevent PP2A(1) binding, cell penetration and apoptosis.In addition the double R77A and R80A mutation known to inactivate the mitochondriotoxic Vpr(71-82) domain, has no effect on the biological properties of the Vpr(77-92) domain.In this context, future studies will be required to determine the functional relevance of the Vpr(77-92) domain in full length Vpr protein and also in entire HIV provirus.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire E3 Phosphatases, Unité Signalisation Moléculaire et Activation Cellulaire, Institut Pasteur, Paris, France.

ABSTRACT

Background: The hallmark of HIV-1 pathogenesis is the progressive CD4(+) T cell depletion and high propensity of CD4(+) T cells to apoptosis. HIV-1 viral protein R (Vpr) is a major pro-apoptotic gene product. A first Vpr-mediated apoptotic mechanism that requires a physical interaction of HIV-1 Vpr(71-82) mitochondriotoxic domain containing the conserved sequence (71-)HFRIGCRHSRIG(-82) with the Adenine Nucleotide Translocator (ANT) has been characterized. The family of Ser/Thr protein phosphatase PP2A interacts with several viral proteins to regulate cell growth and apoptotic pathways. Previous studies based on yeast two hybrid assays and mutational experiments indicated that PP2A(1) is involved in the induction of G2 arrest by HIV-1 Vpr.

Principal findings: Experiments combining pull-down, cell penetration and apoptosis analyses in distinct human cells indicate that the PP2A(1) binding sequence from Vpr(77-92) is a new cell penetrating apoptotic sequence. We also found that the I84P mutation or the IIQ/VTR(83-85) and T89A substitutions in the Vpr(77-92) sequence prevent PP2A(1) binding, cell penetration and apoptosis. In addition the double R77A and R80A mutation known to inactivate the mitochondriotoxic Vpr(71-82) domain, has no effect on the biological properties of the Vpr(77-92) domain.

Conclusion: Together our data provide evidence for the first time that the Vpr(77-92) sequence delineates a biological active domain of Vpr with PP2A(1) binding and pro-apoptotic capacities and, it is conceivable that this cell penetrating sequence may account for the Vpr internalization in uninfected cells. Finally, our data also implicate the existence of two partially overlapping pro-apoptotic domains in the Vpr C-terminal part, a redundancy that represents a new approach to address the question of biological relevance of HIV-1 Vpr. In this context, future studies will be required to determine the functional relevance of the Vpr(77-92) domain in full length Vpr protein and also in entire HIV provirus.

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Effect of DPT-Vpr peptides on cell death.(A) Nuclear morphology illustrated with SK-N-SH cells was evaluated with DAPI (blue) and DNA strand breaks were identified using TUNEL labeling (green). (B) Histograms show percentages of positive SK-N-SH cells (left panel) or HeLa cells (right panel) detected in TUNEL assay.
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pone-0013760-g003: Effect of DPT-Vpr peptides on cell death.(A) Nuclear morphology illustrated with SK-N-SH cells was evaluated with DAPI (blue) and DNA strand breaks were identified using TUNEL labeling (green). (B) Histograms show percentages of positive SK-N-SH cells (left panel) or HeLa cells (right panel) detected in TUNEL assay.

Mentions: Given the critical role of PP2A in the control of apoptotic events and in consistence with the rational of our DPT concept, we hypothesized that DPT-Vpr1 could contain a naturally occurring pro-apoptotic DPT-sequence. To test this possibility, we first used TUNEL assay to monitor apoptosis in adherent HeLa or neuronal SK-N-SH cells. Figure 3A illustrates the high apoptotic level obtained in SK-N-SH cells treated with DPT-Vpr1 and DPT-Vpr3, in contrast, DPT-Vpr2 has no significant effect. This apoptotic effect was quantified in both Hela and SK-N-SH cells and we found that DPT-Vpr1 and DPT-Vpr3 induce apoptosis in 60 to 75% of the cells (Fig. 3B). DPT-Vpr1 and DPT-Vpr3-meditated apoptosis was also assayed in non-adherent Jurkat T cells using Annexin V, an early marker of apoptosis. As indicated in figure 4 upper panel, DPT-Vpr1 and DPT-Vpr3 peptides but not DPT-Vpr2, induced phosphatidylserine translocation. Furthermore we also analyzed a possible drop in mitochondrial membrane potential (ΔΨm), an earlier event in apoptosis. We found that similarly to Vpr71–96, a control peptide containing the mitochondrial death domain from pNL4.3 [11], DPT-Vpr1 and DPT-Vpr3 but not DPT-Vpr2 induced a decrease in ΔΨm (Fig. 4 lower panel). All together these results indicate that the Vpr77–92 sequence from 89.6 isolate is a new PP2A1-binding death domain. Interestingly divergent residues with 89.6 Vpr77–92 sequence are located in the Vpr77–92 domains from a previously published long-term non progressive (LTNP) cohort [12].


PP2A1 binding, cell transducing and apoptotic properties of Vpr(77-92): a new functional domain of HIV-1 Vpr proteins.

Godet AN, Guergnon J, Croset A, Cayla X, Falanga PB, Colle JH, Garcia A - PLoS ONE (2010)

Effect of DPT-Vpr peptides on cell death.(A) Nuclear morphology illustrated with SK-N-SH cells was evaluated with DAPI (blue) and DNA strand breaks were identified using TUNEL labeling (green). (B) Histograms show percentages of positive SK-N-SH cells (left panel) or HeLa cells (right panel) detected in TUNEL assay.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2967473&req=5

pone-0013760-g003: Effect of DPT-Vpr peptides on cell death.(A) Nuclear morphology illustrated with SK-N-SH cells was evaluated with DAPI (blue) and DNA strand breaks were identified using TUNEL labeling (green). (B) Histograms show percentages of positive SK-N-SH cells (left panel) or HeLa cells (right panel) detected in TUNEL assay.
Mentions: Given the critical role of PP2A in the control of apoptotic events and in consistence with the rational of our DPT concept, we hypothesized that DPT-Vpr1 could contain a naturally occurring pro-apoptotic DPT-sequence. To test this possibility, we first used TUNEL assay to monitor apoptosis in adherent HeLa or neuronal SK-N-SH cells. Figure 3A illustrates the high apoptotic level obtained in SK-N-SH cells treated with DPT-Vpr1 and DPT-Vpr3, in contrast, DPT-Vpr2 has no significant effect. This apoptotic effect was quantified in both Hela and SK-N-SH cells and we found that DPT-Vpr1 and DPT-Vpr3 induce apoptosis in 60 to 75% of the cells (Fig. 3B). DPT-Vpr1 and DPT-Vpr3-meditated apoptosis was also assayed in non-adherent Jurkat T cells using Annexin V, an early marker of apoptosis. As indicated in figure 4 upper panel, DPT-Vpr1 and DPT-Vpr3 peptides but not DPT-Vpr2, induced phosphatidylserine translocation. Furthermore we also analyzed a possible drop in mitochondrial membrane potential (ΔΨm), an earlier event in apoptosis. We found that similarly to Vpr71–96, a control peptide containing the mitochondrial death domain from pNL4.3 [11], DPT-Vpr1 and DPT-Vpr3 but not DPT-Vpr2 induced a decrease in ΔΨm (Fig. 4 lower panel). All together these results indicate that the Vpr77–92 sequence from 89.6 isolate is a new PP2A1-binding death domain. Interestingly divergent residues with 89.6 Vpr77–92 sequence are located in the Vpr77–92 domains from a previously published long-term non progressive (LTNP) cohort [12].

Bottom Line: We also found that the I84P mutation or the IIQ/VTR(83-85) and T89A substitutions in the Vpr(77-92) sequence prevent PP2A(1) binding, cell penetration and apoptosis.In addition the double R77A and R80A mutation known to inactivate the mitochondriotoxic Vpr(71-82) domain, has no effect on the biological properties of the Vpr(77-92) domain.In this context, future studies will be required to determine the functional relevance of the Vpr(77-92) domain in full length Vpr protein and also in entire HIV provirus.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire E3 Phosphatases, Unité Signalisation Moléculaire et Activation Cellulaire, Institut Pasteur, Paris, France.

ABSTRACT

Background: The hallmark of HIV-1 pathogenesis is the progressive CD4(+) T cell depletion and high propensity of CD4(+) T cells to apoptosis. HIV-1 viral protein R (Vpr) is a major pro-apoptotic gene product. A first Vpr-mediated apoptotic mechanism that requires a physical interaction of HIV-1 Vpr(71-82) mitochondriotoxic domain containing the conserved sequence (71-)HFRIGCRHSRIG(-82) with the Adenine Nucleotide Translocator (ANT) has been characterized. The family of Ser/Thr protein phosphatase PP2A interacts with several viral proteins to regulate cell growth and apoptotic pathways. Previous studies based on yeast two hybrid assays and mutational experiments indicated that PP2A(1) is involved in the induction of G2 arrest by HIV-1 Vpr.

Principal findings: Experiments combining pull-down, cell penetration and apoptosis analyses in distinct human cells indicate that the PP2A(1) binding sequence from Vpr(77-92) is a new cell penetrating apoptotic sequence. We also found that the I84P mutation or the IIQ/VTR(83-85) and T89A substitutions in the Vpr(77-92) sequence prevent PP2A(1) binding, cell penetration and apoptosis. In addition the double R77A and R80A mutation known to inactivate the mitochondriotoxic Vpr(71-82) domain, has no effect on the biological properties of the Vpr(77-92) domain.

Conclusion: Together our data provide evidence for the first time that the Vpr(77-92) sequence delineates a biological active domain of Vpr with PP2A(1) binding and pro-apoptotic capacities and, it is conceivable that this cell penetrating sequence may account for the Vpr internalization in uninfected cells. Finally, our data also implicate the existence of two partially overlapping pro-apoptotic domains in the Vpr C-terminal part, a redundancy that represents a new approach to address the question of biological relevance of HIV-1 Vpr. In this context, future studies will be required to determine the functional relevance of the Vpr(77-92) domain in full length Vpr protein and also in entire HIV provirus.

Show MeSH
Related in: MedlinePlus